Of all the proteins in human urine, Tamm-Horsfall protein (abbreviated as THP; also named uromodulin) is by far the most abundant. THP is made by kidneys' functionally specialized epithelial cells comprising the thick ascending limb of loop o Henle. Despite its abundance, kidney-specificity and evolutionary conservation, questions about THP's function(s) remain. During the last funding period, we have made major progress in understanding not only the in vivo biological functions of THP, but also its involvement in kidney diseases. Our approach has been to generate, using genetic engineering, knockout (KO) mice deficient for THP or transgenic mice expressing a point mutation of THP. We found that the THP KO mice are highly prone to experimental urinary tract infections by type 1-fimbriated E. coli; that they spontaneously develop intra-renal calcification consisting of calcium phosphate in the interstitial space of the renal papillae; and that they are more susceptible to experimental acute kidney injury than their wild-type counterparts. Additionally, we found that the transgenic mice expressing a THP mutation exhibit marked defects in tubular function and hyperuricemia. Importantly, the intra-renal calcification and hyperuricemia observed in our mouse models bear striking resemblances to certain types of idiopathic kidney stones and hereditary hyperuricemic nephropathies in humans, respectively. The main goal of this renewal proposal is to significantly expand and deepen our understanding of the molecular and cellular mechanisms whereby defects of THP lead to human-relevant disease conditions. Specifically, we will investigate how interstitial calcification in THP KO mice originate and evolve in a spatial and temporal manner by performing ultra-structural and chemical and protein composition analyses. We will examine how renal epithelial cells uptake the intratubular crystals and how this leads to cytotoxicity. We will determine whether formation of bona-fide kidney stones in THP KO mice relies on urinary super saturation of calcium phosphate or calcium oxalate by generating compound, genetically engineered mice that naturally develop these conditions. Finally, we will determine the in vivo effects of specific chemical chaperones in relieving the pathological effects of human-relevant THP mutation, utilizing the transgenic models we recently generated. Together, these four interconnected series of studies should have a major impact on understanding the biological functions and disease contributions of THP and offer insights into how THP-associated kidney diseases can be better managed clinically.